Hypotensors

ABSTRACT

There are provided a method for selection of a substance which is capable of controlling activation of prorenin where an adjusting ability of the activation of prorenin by protein-protein interaction in a profragment region of prorenin as indicator is used; a prorenin activation controlling substance having a function of controlling the activation of prorenin based on protein-protein interaction by a profragment region of prorenin; and hypotensor, organ hypertrophy suppressor and arterial thickening suppressor containing the prorenin activation controlling substance as an effective ingredient.

This application claims the benefit of earlier filed InternationalApplication No. PCT/JP01/03034 filed Apr. 9, 2001.

TECHNICAL FIELD

The present invention relates to hypotensors, organ hypertrophysuppressors and arterial thickening suppressors and also to a method forselecting the same.

BACKGROUND ART

It has been already known that a renin-angiotensin-aldosterone systemparticipates in a rise of blood pressure (Tokyo Joshi Idar Zasshi,60(4): 342˜350, 1999) Actually, angiotensinogen secreted from liver isconverted to angiotensin I (hereinafter, referred to as AI) by theaction of renin derived from kidney and is further converted toangiotensin II (hereinafter, referred to as AII) by the action ofangiotensin converting enzyme (ACE). AII directly acts on blood vesselto cause vasoconstriction and also acts on adrenal cortex to promotebiosynthesis and secretion of aldosterone and to cause retention of Naand water whereupon hypertension is resulted.

Therefore, as a therapeutic method for hypertension, pharmaceuticalagents acting on a renin-angiotensin system have been positioned ashypotensors of the newest type at present and ACE inhibitors have beenpractically used. At the same time, renin inhibitors have been studiedas hypotensors but, due to their side effects, their development hasbeen given up. ACE inhibitors are specific inhibitors for angiotensinconverting enzyme and captopril (generic name) is well known. However,ACE inhibitors have side effects such as dry cough and do not exhibit aclinical efficacy to the extent of an inhibitory effect on the enzymaticactivity in vitro and, therefore, a combination therapy with arenin-angiotensin system inhibitor having different action mechanism hasbeen attempted. Accordingly, there has been a strong demand for highlyeffective hypotensors, blood vessel thickening suppressors and organhypertrophy suppressors which are other than the hypotensors having beenreported or used already.

Renin is biosynthesized mostly in kidney as pre-prorenin comprising 406amino acids that is a precursor thereof whose 23 amino acids atN-terminal are cleaved to give prorenin and then 43 amino acids arefurther cleaved from N-terminal to give renin comprising 340 aminoacids. Renin is a proteinase by which angiotensinogen is specificallyhydrolyzed to produce AI. However, prorenin which is a precursor ofrenin does not usually exhibit such an enzymatic activity. Therefore,although the amount of prorenin existing in blood is about ten-fold ofthat of renin, it has been believed that the active substance in arenin-angiotensin system is renin or renin that is produced byhydrolysis of prorenin.

The present inventors previously found that, when prorenin which is aninactive renin precursor forms an immune complex in vitro as it combineswith an antibody which specifically recognizes a fragment of 43 aminoacid (hereinafter, referred to as “profragment” or “pf”) at N-terminalwhich is cleaved upon at the time of production of renin from prorenin,a protein function or, in other words, an enzymatic activity (reninactivity) is expressed in a non-enzymatic manner under a physiologicalcondition without alteration in the primary structure (Japanese PatentLaid-Open No. H10-279,600 and U.S. Pat. No. 5,945,512), using ananti-human prorenin pf antibody. With regard to a means for activationof prorenin, there have been known methods of conversion to renin usingprotease, a method where activation is carried out without alteration inthe primary structure under an acidic condition and a low-temperature(Nature, 288, 702-705, 1980), (J. Biol. Chem., 262, 2472-2477, 1987),(Clin. Chem., 37, 1811-1819, 1991), (J. Biol. Chem., 267, 11753-11759,1992), and a method where a low-molecular renin inhibitor is combined toan enzymatically active portion buried in the deep grooves of thethree-dimensional structure of prorenin (J. Biol. Chem., 267,22837-22842, 1992) to convert into an open type, etc. However, mechanismfor the activation of prorenin in vivo and its function have still beenambiguous.

SUMMARY OF THE INVENTION

Taking the above-mentioned circumstances into consideration, the presentinventors have carried out an investigation and found that, when ananti-pf antibody which is able to activate prorenin or a partial peptidederived from prorenin profragment which is an antigen for the saidantibody is administered to a pathological animal model of hypertension,there are achieved hypotensive action, organ hypertrophy suppressingaction and arterial thickening suppressing action. Thus, it has beenfound that, when a protein-protein interaction in a profragment regionof prorenin is inhibited, activation of prorenin is suppressed in vivoand thereby it has been firstly proved that activation of prorenin takesplace in vivo. On the basis of such a finding, it has been found that,when a substance that inhibits the activation of prorenin in vivo isused, provided are hypotensors, organ hypertrophy suppressors andarterial thickening suppressors and also a method for selection thereofwhereupon the present invention has been achieved.

Thus, embodiments of the present invention are as follows.

1) A method for selection of a substance capable of controllingactivation of prorenin in which the method is carried out using, as anindicator, an adjusting ability of the activation of prorenin whichoccurs by protein-protein interaction in the profragment region ofprorenin without alteration in a primary structure;

2) A method for selection of a substance capable of controllingactivation of prorenin in which the method is carried out using, as anindicator, an adjusting ability of the activation of prorenin by anantibody against the profragment region of prorenin;

3) The method for selection according to the above 1) or 2), wherein thesubstance to be selected is a substance which is designed on the basisof information of at least three amino acid sequences of the amino acidsequence in the profragment region of prorenin;

4) The method for selection according to the above 1) or 2), wherein thesubstance to be selected is a substance which is designed on the basisof information of at least three amino acid sequences in from the firstto the 19th or from 27th to the 41st amino acid sequence from N-terminalof the amino acid sequence in the profragment region of prorenin;

5) The method for selection according to the above 1) or 2), wherein thesubstance to be selected is a substance which is designed on the basisof information of at least three amino acid sequences in from the 5th tothe 19th amino acid sequence from N-terminal of the amino acid sequencein the profragment region of prorenin;

6) The method for selection according to any of the above 3) to 5),wherein the substance to be selected is a peptide;

7) The method for the selection according to any of the above 3) to 5),wherein the substance to be selected is a low-molecular compound;

8) The method for selection according to any of the above 1) to 7),wherein the prorenin is human prorenin;

9) The method for selection according to any of the above 1) to 7),wherein the prorenin is rat prorenin;

10) A prorenin activation controlling substance having a function ofcontrolling activation of prorenin which occurs by protein-proteininteraction in the profragment region of prorenin without alteration ina primary structure;

11) a function of controlling activation of prorenin which occurs byprotein-protein interaction in the profragment region of proreninwithout alteration in a primary structure, wherein the substance isselected by any of the methods mentioned in the above 1) to 9);

12) A prorenin activation controlling substance having a function ofinhibiting the activation of prorenin without a primary structuralchange occurred by a protein-protein interaction in the profragmentregion of prorenin;

13) A prorenin activation controlling substance having a function ofinhibiting activation of prorenin which occurs by protein-proteininteraction in the profragment region of prorenin without alteration ina primary structure and retaining no renin-inhibiting activity;

14) A prorenin activation controlling substance having a function ofinhibiting activation of prorenin which occurs by protein-proteininteraction in the profragment region of prorenin without alteration ina primary structure, wherein the substance is selected by any of themethods mentioned in the above 1) to 9);

15) A prorenin activation controlling substance having a function ofinhibiting activation of prorenin which occurs by protein-proteininteraction in the profragment region of prorenin without alteration ina primary structure and retaining no renin-inhibiting activity, which isselected by any of the methods mentioned in the above 1) to 9);

16) A prorenin activation controlling substance comprising a peptidehaving a partial sequence selected from amino acid sequence ofprofragment region of prorenin or an equivalent peptide thereto andhaving a function of inhibiting activation of prorenin which occurs byprotein-protein interaction in the profragment region of proreninwithout alteration in a primary structure;

17) A prorenin activation controlling substance comprising a peptidehaving a continued 3 to 10 partial sequence selected from amino acidsequence of profragment region of prorenin and having a function ofinhibiting activation of prorenin which occurs by protein-proteininteraction in the profragment region of prorenin without alteration ina primary structure;

18) A prorenin activation controlling substance comprising a peptidehaving a continued 3 to 8 partial sequence selected from amino acidsequence of profragment region of prorenin and having a function ofinhibiting activation of prorenin which occurs by protein-proteininteraction in the profragment region of prorenin without alteration ina primary structure;

19) A prorenin activation controlling substance comprising a peptidehaving a continued 3 to 6 partial sequence selected from amino acidsequence of profragment region of prorenin and having a function ofinhibiting activation of prorenin which occurs by protein-proteininteraction in the profragment region of prorenin without alteration ina primary structure;

20) A prorenin activation controlling substance comprising a peptideselected from the peptide mentioned in SEQ ID NO: 7 to NO: 14 of theSequence Listing and having a function of inhibiting activation ofprorenin which occurs by protein-protein interaction in the profragmentregion of prorenin without alteration in a primary structure;

21) A prorenin activation controlling substance comprising a peptideselected from the peptide mentioned in SEQ ID NO: 3, NO: 4 and from NO:15 to NO: 20 of the Sequence Listing and having a function of inhibitingactivation of prorenin which occurs by protein-protein interaction inthe profragment region of prorenin without alteration in a primarystructure;

22) A prorenin activation controlling substance having a function ofinhibiting activation of prorenin which occurs by protein-proteininteraction in the profragment region of prorenin without alteration ina primary structure, wherein the substance is a low-molecular compounddesigned from the information of amino acid sequence of profragmentregion of prorenin;

23) The prorenin activation controlling substance according to any ofthe above 16) to 22), wherein the substance has a function of inhibitingactivation of prorenin which occurs by protein-protein interaction inthe profragment region of prorenin without alteration in a primarystructure and retains no renin-inhibiting activity;

24) The prorenin activation controlling substance according to any ofthe above 12) to 23), wherein an effect of inhibiting activation ofprorenin is caused by an antagonistic action against the protein-proteininteraction in the profragment region of prorenin;

25) A prorenin activation controlling substance which is characterizedin that activation of prorenin is inhibited in vivo by removal ofprorenin based on an antigen-antibody reaction to the profragment regionof prorenin;

26) A prorenin activation controlling substance comprising an antibodyagainst the profragment region of prorenin which is capable ofinhibiting the prorenin activation in vivo by removal of prorenin basedon an antigen-antibody reaction to the profragment region of proreninantigen-antibody reaction to the profragment region of prorenin;

27). A hypotensor containing at least one of the prorenin activationcontrolling substances mentioned in any of the above 12) to 26) as aneffective ingredient;

28) The hypotensor according to the above 27), wherein about 10% or morehypotensive rate is available as a hypotensive effect at least at the12th hour after administration in vivo;

29) The hypotensor according to the above 27), wherein a hypotensiveeffect is selectively achieved only in patients suffering fromhypertension;

30) An organ hypertrophy suppressor containing at least one of theprorenin activation controlling substances mentioned in any of the above12) to 26);

31) The organ hypertrophy suppressor according to the above 30), whereinan organ hypertrophy can be suppressed in heart and/or kidney;

32) An arterial thickening suppressor containing at least one of theprorenin activation controlling substances mentioned in any of the above12) to 26) as an effective ingredient;

33) A method for a treatment of hypertension using any of thehypotensors mentioned in the above 27) to 29);

34) A method for a treatment of cardiac insufficiency or renalinsufficiency using the organ hypertrophy suppressor mentioned in theabove 30) or 31); and

35) A method for a treatment of diseases accompanied by thickening ofblood vessel using the arterial thickening suppressor mentioned in theabove 32).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will be illustrated in detail as hereunder and themeanings commonly understood by one of ordinary skill in the art towhich the present invention pertains, unless otherwise defined. In thepresent specification, reference is made herein to various methodologiesknown to those of ordinary skill in the art. Reference documents such aspublications setting forth such known methodologies to which referenceis made are incorporated herein by reference in their entireties asthough set forth in full.

(Effect of Inhibition of Activation of Prorenin in Vivo)

The present inventors have previously found using an anti-human pfantibody that prorenin can be activated to an open-type structure invitro exhibiting an enzymatic activity without alteration in its primarystructure by a specific antibody to an amino acid fragment of pf regionof prorenin (Japanese Patent Laid-Open No. H10-279,600; U.S. Pat. No.5,945,512). On the basis of such a finding, there was prepared anantibody (anti-rat pf antibody) which specifically recognizes a rat pfpeptide that is in the same site as the peptide (human pf peptide) of pfregion of human prorenin in view of the order of amino acid sequence inthe primary structure of rat prorenin and the said antibody wasadministered to spontaneously hypertensive rats (SHR) which are thepathological animal model for hypertension. As a result, although thesaid anti-rat pf antibody was able to activate the rat prorenin in vitrowithout alteration in its primary structure, it showed a continuoushypotensive effect in vivo as shown in Experimental Examples. Ingeneral, antibody forms an antigen-antibody complex by combining with anantigen in blood and then the said resulting antigen-antibody complex isremoved from the blood by an immune system. Thus, it can be consideredthat a hypotensive effect is achieved due to removal of prorenin oractivated prorenin by combining with an anti-pf antibody. Alternatively,it is conceivable that a hypotensive effect is achieved due toinhibition of combining of prorenin or activated prorenin with itsaction site as a result of combining of the anti-pf antibody withprorenin or activated prorenin in blood.

In addition, the present inventors synthesized a partial peptide derivedfrom rat prorenin profragment which is an antigen against theabove-mentioned anti-rat pf antibody which is able to activate the ratprorenin in vitro and administered it to SHR. Such a peptide is able tocompetitively inhibit the activation of rat prorenin in vitro by ananti-rat pf antibody like the fact that a human pf peptide inhibits theactivation of human prorenin in vitro by an anti-human pf antibody.Thus, such a peptide is able to inhibit the activation of rat proreninthat is resulted by a protein-protein interaction where alteration ofthe primary structure is not accompanied but that of the higher-orderstructure is accompanied. When such a peptide was administered to SHR, asufficient hypotensive effect was achieved at the 12th hour after theadministration as will be mentioned later. However, when such a peptidewas administered to normal rats, there was no variation in bloodpressure. As shown in the Examples which will be mentioned later, thebehavior of such a peptide in the course of time was different from thatof a renin inhibitor H6137 (manufactured by Sigma) or a neutralizedantibody of renin and its hypotensive effect was observed during a longperiod, so that the hypotensive effect by such a peptide was revealed tobe not due to inhibition of renin. In addition, it was also proved that,when such a peptide was continuously administered for 14 days to SHR,hypertrophy of heart and kidney was suppressed and arterial thickening,i.e. blood vessel remodeling, was suppressed as well. Those effectsachieved by administration of the above-mentioned peptide are presumedto be achieved by the fact that the said peptide inhibits the activationof rat prorenin with alteration of the structure accompanied byprotein-protein interaction in vivo or inhibits rat prorenin oractivated rat prorenin to combine with its acting site.

From the above results, it is probable that, in SHR, prorenin exhibitsits enzymatic activity by interacting with prorenin-binding protein toactivate renin-angiotensin system followed by causing the hypertensionand it has been revealed that the pf peptide and anti-pf peptideantibody showed a hypotensive action against hypertension caused byactivation of the renin-angiotensin system. Participation of prorenin inhypertension and prorenin activation in vivo by a protein-proteininteraction in profragment region of prorenin as such has now beenconfirmed for the first time. It has been also found that a proreninactivation controlling substance, which is capable of inhibiting theactivation of prorenin with alteration in an structure due toprotein-protein interaction and the protein-protein interaction ofprorenin or activated prorenin with its acting site, is useful ashypotensor, organ hypertrophy suppressor, arterial thickeningsuppressor, etc.

The number of amino acid residues of the pf peptide of rat prorenin isthe same as that of human prorenin pf peptide but the amino acidsconstituting them are different. In spite of that, as mentioned above, arat pf peptide, which is in the same site as the human pf peptide inview of the order of amino acid sequence in the primary structure thatis an epitope peptide of anti-human pf antibody capable of activatingthe human prorenin in vitro, and a specific antibody against the said pfpeptide showed a hypotensive effect in pathological model rats ofhypertension Accordingly, there is no difference among the species inpharmacological action of prorenin participating in hypertension,structural site concerning the activation of prorenin, and mechanism ofits activation. Thus, although the hypotensive effect of theabove-mentioned peptide was observed in pathological model rats ofhypertension, it goes without saying that the same effect can beexpected for human hypertension as well by selecting an appropriatepeptide in consideration of the amino acid sequence of pf peptide ofhuman prorenin.

Further, as mentioned above, when an inactive enzyme precursor interactswith its specific anti-pf peptide antibody in vitro to express theenzymatic activity or, in other words, protein function, then itsmechanism is conserved over the species, so that pf peptide, antibodyagainst the said pf peptide, pathological animal model, and so on areuseful as a screening method for selecting the therapeutic medicamentfor the symptoms caused by expression of the protein function.

(Method for Selecting a Prorenin Activation Controlling Substance)

A method for the selection of a substance that is capable of controllingthe activation of prorenin according to the present invention ischaracterized in using an adjusting ability of the activation ofprorenin by the protein-protein interaction in a profragment region ofprorenin as an indicator. Here, activation of prorenin by aprotein-protein interaction in a profragment region of prorenin means toopen an enzymatically active site of prorenin which is not accompaniedby the alteration in a primary structure but accompanied by thealteration in a higher-order structure caused by protein-proteininteraction in the pf region. Accordingly, although prorenin which isactivated by the said protein-protein interaction retains the same aminoacid sequence as that of the primary structure of prorenin before beingactivated, it is able to show a renin-like enzymatic activity. Examplesof the protein having a protein-protein interaction in the pf region areantibody which is activated by recognizing the pf peptide, protein whichis an action site of prorenin or activated prorenin in vivo, and proteinin blood which is capable of activating the prorenin by combiningtherewith.

The above-mentioned method for selecting a prorenin activationcontrolling substance can be constructed by utilizing a screening systemfor pharmaceutical agents that has been known per se. For example, whenprorenin and an anti-pf antibody which is capable of activating theprorenin are used to measure the binding of prorenin to the said anti-pfantibody, it is possible to select a substance which inhibits theactivation of prorenin. Further, for example, when prorenin and ananti-pf antibody which is capable of activating the prorenin are used tomeasure the enzymatic activity of the activated prorenin, it is possibleto select a substance which inhibits the activation of prorenin or asubstance which inhibits the enzymatic activity of prorenin. Needless tosay, methods for the selection are not limited thereto. In addition, asmentioned above, the amino acids that constitute the amino acid sequenceof pf region of prorenin vary depending upon the species and, therefore,in the selection of a controlling substance for activation of humanprorenin, the use of human prorenin and an antibody against pf peptideof the human prorenin is one of the preferred embodiments.

As an example in the concrete, it is possible to construct a screeningsystem utilizing human prorenin and an antibody that is capable ofactivating the human prorenin by specifically recognizing human proreninpf peptide prepared according to the description in Japanese PatentLaid-Open No. H10-279,600 and U.S. Pat. No. 5,945,512.

With regard to a substance which is an object to be selected in theabove-mentioned selecting method for a prorenin activation controllingsubstance, candidate substances can be generally utilized as objects forthe selection, which are used in a pharmaceutical screening system thathas been known per se, such as low-molecular weight compounds, compoundsderived from natural substances, peptides, and so on.

Low-molecular weight compounds and peptides that are designed based onamino acid sequence information of pf region of prorenin or,particularly, human prorenin may be used as objects for the selection aswell.

Amino acid sequence of pf region of human prorenin has been alreadyknown and comprises 43 amino acids (SEQ ID NO: 1 of the SequenceListing) as shown in Japanese Patent Laid-Open No. 8-285,852. The aminoacid sequence of pf peptide of prorenin is useful information fordesigning a substance which is an object for selecting a proreninactivation controlling substance. Particularly, the information of atleast 3 consecutive or, more preferably, at least 4 consecutive aminoacid sequences in the amino acid sequence that is from the first to the19th (pf 1-19), especially from the 5th to the 19th (pf 5-19) and fromthe 27th to the 41st (pf 27-41) from N-terminal of pf region of proreninis useful for designing the prorenin activation controlling substance.Amino acid sequences of human-derived pf 1-19, human-derived pf 5-19,human derived pf 27-41, rat-derived pf 1-19, rat-derived pf 5-19 andrat-derived pf 27-41 are shown in SEQ ID NO: 2, NO: 3, NO: 4, NO: 6, NO:7 and NO: 8, respectively. Hereinafter, a peptide comprising themoieties from the M-th to the N-th ones from N-terminal of pf regionwill be referred to as “pf M-N”.

As mentioned above, in the amino acid sequence of a prorenin pf region,although numbers of the amino acid residues are same even when thespecies are different, the constituent amino acids are different. InExperimental Examples, the amino acid sequence (SEQ ID NO: 5 of theSequence Listing) of pf peptide of rat prorenin was exemplified as theinformation useful for designing the substance that is an object for theselection of prorenin activation controlling substance. In the Examples,fragments of the amino acid sequence of rat prorenin pf region wassynthesized, such as pf 1-11 (SEQ ID NO: 9 of Sequence Listing), pf 5-19(SEQ ID NO: 7 of Sequence Listing), pf 1-4 (SEQ ID NO: 10 of SequenceListing), pf 1-7 (SEQ ID NO: 11 of Sequence Listing), pf 5-11 (SEQ IDNO: 12 of Sequence Listing), pf 12-19 (SEQ ID NO: 13 of SequenceListing), pf 11-15 (SEQ ID NO: 14 of Sequence Listing) and pf 27-41 (SEQID NO: 8 of Sequence Listing), followed by their hypotensive effect,organ hypertrophy suppressing effect and arterial thickening suppressingeffect due to suppression of prorenin activation thereby. However, suchsequences are not limited thereto. Anyone ordinary skilled in the art isable to synthesize appropriately at least 3 peptides by a method whichis known per se and to design and synthesize a substance, which is ableto be an object to be selected, based on the peptide synthesized assuch.

Further, as mentioned above, a specific antibody to rat pf peptide thatis in the same site in view of the order of amino acid sequence in theprimary structure as the human pf peptide which is an epitope of ananti-human pf antibody which is able to activate human prorenin in vitroshowed a hypotensive action in a hypertensive pathological model ratsand, therefore, it is easily presumed that the human prorenin pf peptidewhere the amino acid sequence order in the primary structure is same asthe rat pf peptide shows the similar effect to human being as well. Asone of the preferred embodiments, the pf peptide of human prorenin isused as the information for designing a substance that is to be anobject for the selection.

The substance which is to be an object for the selection, when it is apeptide, is able to be appropriately designed on the basis ofinformation of amino acid sequence in the prorenin pf region asmentioned above and provided for the selection. The amino acidconsisting the said peptide are not always necessary to be same as theamino acid sequence of the prorenin region but that may be a peptidewhere mutation such as deficiency, substitution, addition and insertionmay be introduced into the amino acid sequence of the said peptide sofar as the same function as the said peptide is available (hereinafter,that may sometimes be called “equivalent peptide”).

The substance that is to be an object for the selection may also be alow-molecular weight compound that is obtained by drug design based onstructural complementarity according to the information on the basis ofthe secondary and/or tertiary structure of amino acid sequence of pfregion.

As a result of the above-mentioned method, a substance is obtained whichis able to inhibit the activation of human prorenin by anti-human pfpeptide antibody. From the substances obtained, it is also possible toselect the substances having hypotensive effect, organ hypertrophysuppressing effect and arterial thickening suppressing effect, byaccumulating the information based on peptide having a partial sequenceselected from the amino acid sequence of pf region, known hypotensors,and the like or carrying out an experiments using an animal model and soon.

(A Prorenin Activation Controlling Substance)

Thus, the present invention further provides a prorenin activationcontrolling substance that is capable of controlling the activation ofprorenin due to the protein-protein interaction in the pf region ofprorenin.

One of the embodiments of the present invention is a prorenin activationcontrolling substance that is capable of controlling the activation ofhuman prorenin.

The prorenin activation controlling substance according to the presentinvention may be a substance which competitively inhibits theprotein-protein interaction that leads to prorenin activation, asubstance which antagonistically inhibits the binding of prorenin oractivated prorenin at the site on which they act, a substance which hasantigen-antibody reactivity to prorenin or activated prorenin, asubstance which acts on the site on which prorenin or activated proreninact to inhibit the action of prorenin or activated prorenin, and thelike.

One of the preferred embodiments is that the above-mentioned proreninactivation controlling substance is the one that has a function ofinhibiting the prorenin activation based on the protein-proteininteraction in the pf region of prorenin and has no renin-inhibitingactivity. Thus, the above-mentioned prorenin activation controllingsubstance has no direct inhibiting action on renin as its actionmechanism. Therefore, unlike renin inhibitors, it is able to inhibit theactivity of prorenin that is activated in a pathological state withoutinhibiting a renin-angiotensin system that has an important role in thehomeostatic mechanism of life. That is an important characteristic ofthe prorenin activation controlling substance according to the presentinvention.

In the concrete, a peptide having a partial sequence selected from theamino acid sequence of pf region of prorenin, an equivalent peptide tothe said peptide, a low-molecular weight substance designed from theinformation of the amino acid sequence of pf region of prorenin, anantibody against the pf region of prorenin, and so on are exemplified,although they are not limited thereto.

One of the preferred embodiments is that the above-mentioned peptide isa peptide having a partial sequence selected from the amino acidsequence of pf region of human prorenin or an equivalent peptidethereto. For example, with regard to a peptide derived from the aminoacid sequence of pf region of human prorenin, the following peptides maybe exemplified.

hp 1: human prorenin pf 1-11 (SEQ ID NO: 15 of the Sequence Listing)

hp 2: human prorenin pf 5-11 (SEQ ID NO: 16 of the Sequence Listing)

hp 3: human prorenin pf 5-19 (SEQ ID NO: 3 of the Sequence Listing)

hp 4: human prorenin pf 1-4 (SEQ ID NO: 17 of the Sequence Listing)

hp 5: human prorenin pf 1-7 (SEQ ID NO: 18 of the Sequence Listing)

hp 6: human prorenin pf 12-19 (SEQ ID NO: 19 of the Sequence Listing)

hp 7: human prorenin pf 11-15 (SEQ ID NO: 20 of the Sequence Listing)and

hp 8: human prorenin pf 27-41 (SEQ ID NO: 4 of the Sequence Listing)

In controlling the activation of prorenin, one of those peptides may beused or two or more thereof may be mixed to use.

As one of the preferred embodiments, the above-mentioned antibody is anantibody against the human prorenin pf region. For example, anti-humanpf antibodies may be exemplified, which are obtained by using thefollowing peptides derived from the amino acid sequence of humanprorenin pf region as antigens.

hp 9: human prorenin pf 1-15 (SEQ ID NO: 21 of the Sequence Listing)

hp 10: human prorenin pf 18-30 (SEQ ID NO: 22 of the Sequence Listing)and

hp 11: human prorenin pf 30-41 (SEQ ID NO: 23 of the Sequence Listing)

In controlling the activation of prorenin, only one kind of antibody maybe used or two or more kinds thereof may be mixed to use. When theabove-mentioned antibody is used in pharmaceuticals as a proreninactivation controlling substance, the said antibody is preferred to be amonoclonal antibody that can be obtained by a known method and is alsopreferred to be prepared as a human type antibody. As to a method forthe preparation of human type antibody, it is possible to utilize amethod known per se (J. Immunol. Methods, 100, 5-40, 1987).

(Hypotensors)

As one of the embodiments, the present invention provides a hypotensorcontaining at least one kind of the above-mentioned prorenin activationcontrolling substances as an effective ingredient. The said hypotensoris characterized in that, at even 12 hours later from its administrationinto living body, it still achieves a sufficient hypotensive action andthe duration of the effect is quite long. For example, in an experimentin pathological model rats of hypertension, a hypotensive rate of atleast around 10% as SBP (systolic blood pressure) values (%) was shownafter 12 hours from the administration. Although that value is not sohigh as hypotensors, one of ordinary skill in the art is easily able togive higher effect by improving dose, administration period and/oradministering method, and so on. On the other hand, the in vivo effectof renin inhibitors, which are the hypotensors being available hitherto,appears immediately after the administration and, after that, itdisappears within minutes. Further, the in vivo effect of ACE inhibitorscontinues for 6-8 hours after the administration, though it disappearsafter that. Consequently, the sustained hypotensive effect of thehypotensors according to the present invention for long time is a veryuseful characteristic when improvement of hypertension is taken intoconsideration.

Further, the hypotensors according to the present invention shows ahypotensive effect by a mechanism which is different from that ofremedies for hypertension that have been available hitherto such as ACEinhibitors and renin inhibitors and they may be used in a treatment ofhypertension together with the remedies for hypertension and the likethat have been already available.

Furthermore, the above-mentioned hypotensors are able to achieve theirhypotensive effect selectively only to the patients suffering fromhypertension. In the experiment comparing normal rats with pathologicalmodel rats of hypertension, it was proved that the above-mentionedhypotensors acted on pathological model rats of hypertension only. Thus,the hypotensor according to the present invention is useful for thetreatment of hypertension.

(Organ Hypertrophy Suppressors)

The present invention further provides a novel suppressor forhypertrophy of organs containing at least one kind of theabove-mentioned prorenin activation controlling substances as aneffective ingredient. The organ hypertrophy suppressor according to thepresent invention is able to suppress the organ hypertrophy in heartand/or kidney. Accordingly, the said organ hypertrophy suppressor can beexpected to have an effect in cardiac insufficiency and renalinsufficiency.

(Arterial Thickening Suppressors)

The present invention provides a novel suppressor for thickening ofartery containing at least one kind of the above-mentioned proreninactivation suppressing substances as an effective ingredient. Thearterial thickening suppressor according to the present invention isable to suppress the thickening, that is to say remodeling of bloodvessel, of pulmonary artery, femoral artery, and so on. Accordingly, thesaid arterial thickening suppressor is effective for the diseasesaccompanied by thickening of blood vessel that is to say remodeling ofblood vessel, such as arteriosclerosis.

(Making into Pharmaceutical Preparations)

In making the above-mentioned hypotensors, organ hypertrophy suppressorsand arterial thickening suppressors into pharmaceutical preparations,known methods may be appropriately used depending upon the physicalproperty of the peptide or the low-molecular weight compound. Forexample, methods for manufacturing tablets, capsules, aqueous solutionpreparations, ethanolic solution preparations, liposome preparations,fatty emulsions, inclusion compounds including cyclodextrin or the like,and so on, may be utilized.

Powder, pills, capsules and tablets may be manufactured using excipientsuch as lactose, glucose, sucrose and mannitol; disintegrating agentsuch as starch and sodium alginate; lubricant such as magnesium stearateand talc; binder such as polyvinyl alcohol, hydroxypropyl cellulose andgelatin; surface-active agent such as fatty acid ester; plasticizer suchas glycerol; etc. In the manufacture of tablets and capsules, a solidpharmaceutical carrier is used.

Suspension may be manufactured by using water; saccharide such assucrose, sorbitol and fructose; glycols such as PEG; and oils.

Solutions for injection can be prepared using a carrier comprising asalt solution, a glucose solution or a mixture of salt water and aglucose solution.

Manufacture of liposome preparation may be for example carried out insuch a manner that a solution where the said substance is dissolved in asolvent (such as ethanol) is added to a solution where phospholipid isdissolved in an organic solvent (such as chloroform), and the solventsare evaporated therefrom, and then a phosphate buffer is added theretofollowed by subjecting to shaking, ultrasonic treatment and centrifugalseparation and the supernatant is filtered to recover.

Manufacture of a fatty emulsion may be for example carried out in such amanner that the said substance, oil component (such as soybean oil,sesame oil, olive oil and vegetable oil, MCT, and the like.), emulsifier(such as phospholipid), etc. are mixed and heated and a necessary amountof water is added to the resulting solution followed by subjecting toemulsifying/homogenizing treatment using an emulsifier (homogenizer suchas that of a high-pressure type, a ultrasonic type, etc.). It is alsopossible that the product is freeze-dried. In the manufacture of a fattyemulsion, an emulsifying aid may be used and glycerol and saccharides(such as glucose, sorbitol, fructose and the like) are exemplified asthe emulsifying aid.

Manufacture of an inclusion compound with cyclodextrin may be carriedout for example in such a manner that a solution where cyclodextrin isdissolved in water or the like by heating is added to a solution wherethe said substance is dissolved in a solvent (such as ethanol), and themixture is cooled to separate precipitate followed by filtering anddrying with sterilization. With regard to the cyclodextrin used there,cyclodextrin having different pore diameters (α, β and γ types) may beappropriately selected depending upon the size of the said substance.

Dose of the above-mentioned hypotensor, organ hypertrophy suppressor orarterial thickening suppressor may be appropriately selected dependingupon symptom, sex, age and body weight of the patient. Route for theadministration may be either of oral and parenteral. One of thepreferred embodiments is that an orally applicable preparation ismanufactured and is administered per os. An examples of the dose isaround 1˜1,000 μg per day.

EXAMPLES

The present invention will now be more specifically illustrated by wayof the following examples although the present invention is not limitedthereto but can be variously modified within a range of not departingfrom the technical idea of the present invention.

Example 1

(Preparation of Antibody Recognizing a Peptide Derived from Pf Region ofRat Prorenin)

Pf 1-15 (SEQ ID NO: 24 of the Sequence Listing), pf 18-30 (SEQ ID NO: 25of the Sequence Listing) and pf 30-41 (SEQ ID NO: 26 of the SequenceListing), which are pf peptides of rat prorenin, were prepared by asolid phase method in a conventional manner and, using thoseoligopeptides as antigens, antibodies against them—Ab 1, Ab 2 and Ab3—were prepared according to the descriptions of Examples of JapanesePatent Laid-Open No. H10-279,600 and U.S. Pat. No. 5,945,512. To be morespecific, the above-mentioned oligopeptide which was an antigen wasimmunized to rabbit of a New Zealand white species together with anadjuvant to prepare anti-serum. The resulting anti-serum was purifiedaccording to the method mentioned in the patents.

Ab 1: antibody which specifically recognizes pf 1-15

Ab 2: antibody which specifically recognizes pf 18-30

Ab 3: antibody which specifically recognizes pf 30-41

Further, equal amounts of those antibodies were mixed to prepare Ab mix.Furthermore, activation of prorenin by those antibodies in vitro wasinvestigated by the methods mentioned in the patents and it wasconfirmed that those antibodies were able to activate prorenin.

Example 2

(Preparation of Peptide Derived from Pf Region of Rat Prorenin)

The following peptides derived from the sequence of pf region of ratprorenin were synthesized by a solid phase method in a conventionalmanner.

p 1: pf 1-11 (SEQ ID NO: 9 of the Sequence Listing)

p 2: pf 5-19 (SEQ ID NO: 7 of the Sequence Listing)

p 3: pf 27-41 (SEQ ID NO: 8 of the Sequence Listing)

p 1a: pf 1-4 (SEQ ID NO: 10 of the Sequence Listing)

p 1b: pf 1-7 (SEQ ID NO: 11 of the Sequence Listing)

p 1c: pf 5-11 (SEQ ID NO: 12 of the Sequence Listing)

p 2a: pf 12-19 (SEQ ID NO: 13 of the Sequence Listing) and

p 2b: pf 11-15 (SEQ ID NO: 14 of the Sequence Listing)

Further, equal amounts of p 1, p 2 and p 3 were mixed to prepare a Pmix.

Experimental Example 1

(Investigation of Hypotensive Effect by Anti-pf Peptide Antibody and pfPeptide)

A group comprising three SHR male rats (12˜14 weeks age; body weight:258˜310 g) that are pathological model animals of hypertension was used.Ab mix prepared in Example 1, P mix prepared in Example 2 and dissolvedin a physiological saline, and anti-renin neutralizing antibody andcaptopril, which were the drugs used for comparative study, wereintravenously administered at the dose of 0.44 mg/kg, 0.6 mg/kg, 0.12mg/kg and 100 mg/kg, respectively. Measurement of blood pressure wascarried out in such a manner that a cannula inserted into common carotidartery and external carotid artery was connected to a pressuretransducer (P23XL; Gould Electronics) and introduced into a pressureprocessor signal conditioner (Gould Electronics), followed by carryingout measurement every ten minutes until 1 hour from administration ofthe test substance and every one hour thereafter and the data wererecorded on a thermal array recorder (RS 3400; Gould Electronics).

The anti-renin neutralizing antibody that was a drug for comparativestudy did not show any hypotension until 6 hours from the administrationand, at the 6th hour and the 24th hour, tendencies of hypotension of 11%and 14%, respectively, were noted. P mix and Ab mix showed continuoushypotension of 9% at the highest and 7% at the highest, respectivelyafter administration until the completion of the experiment. In the caseof captopril that was a positive control, the maximum hypotension (31%at the highest) was achieved until the 6th hour from the administrationand, at the completion of the experiment at the 24th hour, nohypotensive action was noted.

As a result, it was revealed that P mix and Ab mix showed a hypotensiveeffect with a different course of time as comparing to anti-reninneutralizing antibody and captopril which was an ACE inhibitor. Thus, itwas found that P mix and Ab mix showed a sustaining hypotensive abilityfor a very long period immediately after the administration. Inaddition, it was presumed therefrom that the hypotensive effect of P mixand Ab mix was not due to inhibition of renin activity.

Experimental Example 2

On the basis of the data obtained in Experimental Example 1, a test wascarried out for P mix with an increased dose (2 mg/kg) using five casesof SHR in the same manner as in Experimental Example 1. A physiologicalsaline was used for a control group. As a result, a transienthypotensive action of 11% was noted immediately after administration.Although that recovered after 5 minutes from administration, hypotensiongradually took place again thereafter to an extent of 9% at the highestwhereupon a significant difference was noted as compared with thecontrol group after 1-6 hour(s) from administration. Thus, it wasconfirmed that P mix had a biphasic hypotensive action

Experimental Example 3

A test was carried out for p 1 (pf 1-11), p 2 (pf 5-19) and p 3 (pf27-41) prepared in Example 2 in the same manner as in ExperimentalExample 1 using 2 or 4 cases of SHR. The result was that, in the case ofadministration of 2 mg/kg of p 1, a transient hypertensive action ofaround 6% was noted immediately after administration, that recoveredafter 5 minutes from administration, then a hypotensive gradually tookplace thereafter to an extent of 6% at the highest and a sustainedeffect was confirmed until 24th hour. In the case of administration of10 mg/kg, hypotension of about 7% was noted at 6 hours afteradministration and, until 24th hour, a sustained effect to an extent ofaround 10% at the highest was noted. In the case of administration of 2mg/kg of p 2, a transient hypotension of around 35% was notedimmediately after administration, it recovered at 5 minutes afteradministration and, after that, a sustained hypotensive action wasconfirmed until 24th hour to an extent of around 6% at the highest. Inthe case of administration of 2 mg/kg of p 3, a transient hypertensionof around 14% was noted immediately after administration, that recoveredat 5 minutes after administration and no action was confirmedthereafter. In the case of administration of 10 mg/kg, a hypotension of7% was noted after 2 hours and, until 24th hour, a sustained hypotensiveaction where 11% was the highest was confirmed.

Thus, it was found that a peptide comprising an amino acid sequence offrom the first to the 11th or from the 5th to the 19th from N-terminalof pf region of rat prorenin and a peptide comprising an amino acidsequence of from the 27th to the 41st corresponding to the C-terminalside of pf region of rat prorenin had a sustained hypotensive action.

Such a result also shows that the transient hypotension immediatelyafter administration of P mix is an action of p 2 and the sustainedhypotensive action thereafter is an interactional action of p 1, p 2 andp3.

Experimental Example 4

As a comparative experiment, the same test as in Example 2 was carriedout using five rats having normal blood pressure (WKY rats) of the sameweeks age in place of SHR. Except a transient hypotension immediatelyafter the administration, P mix did not show any hypotension at all.Accordingly, it was confirmed that P mix did not show a sustainedhypotensive action in normal rats.

Experimental Example 5

The same test as in Experimental Example 1 was carried out for each 2cases of SHR using p 1 (pf 1-11), p 1a (pf 1-4), p 1b (pf 1-7), p 1c (pf5-11), p 2a (pf 12-19) and p 2b (pf 11-15) prepared in Example 2 at thedose of 1 mg/kg for p 2a (pf 12-19) and p 2b (pf 11-15) and 10 mg/kg forall other peptides.

In result, p 1 showed a sustained hypotension to an extent of 14% at thehighest until 24 hours after administration. Although p la showed atransient hypertension of 13% immediately after administration, itshowed a sustained hypotension of 13% at the highest until 24 hoursthereafter. Although p 1b showed a transient hypertension of 17%immediately after administration, it showed a sustained hypotension of13% at the highest until 24 hours thereafter. Although p 1c showed atransient hypertension of 21% immediately after administration, itshowed a sustained hypotension of 16% at the highest until 24 hoursthereafter. Although p 2a showed a transient hypertension of 6%immediately after administration, it showed a sustained hypotension of14% at the highest until 24 hours thereafter. The same effect wasachieved by the use of p 2b as well.

As a result, it was found that a peptide derived from an amino acidsequence of from the first to the 19th from N-terminal of the amino acidsequence of pf region of rat prorenin had a sustained hypotensiveaction.

Experimental Example 6

Each of p 2a (pf 12-19) prepared in Example 2 and H 6137 (which is arenin inhibitor; D-His-Pro-Phe-His-Leu-ψ-[CH₂NH]-Leu-Val-Tyr; Sigma) wasused and administered at the dose of 1 mg/kg via a cannula from externalcarotid artery and the changes of blood pressure in a course of timewere measured by the method mentioned in Example 1.

The result was that p 2a showed a sustained hypotension of 11% at thehighest at 5˜8 hours after administration and that effect continued atleast for 12 hours from the administration. On the other hand, althoughH 6137 showed a sustained hypotension of 9% at the highest at 3˜8 hoursafter administration, a tendency of disappearance of the effect wasobserves after 6 hours of the administration and thereafter.

It has been made clear that, although p 2a requires long time until itsaction appears, its action is long lasting, and in the case of H 6137,time until appearance of the action is quick but its acting period isshort. From the difference in the behaviors of the hypotensive effect ina course of time as such, it is presumed that the hypotensive effect ofprorenin activation controlling substance represented by p 2a is not dueto inhibition of renin.

Experimental Example 7

The p 1c (pf 5-11) prepared in Example 2 was intravenously administeredonce daily for consecutive 14 days at the dose of 1.6 mg/kg to a groupcomprising 4 SHRs. On the 14th day, the rats were killed by bleeding,followed by excising heart and kidney to measure their weights. Theresult is shown in Table 1 in terms of weight of the organ per 100 g ofbody weight. As shown in Table 1, the weight of heart and kidney of SHRto which p 1c was administered was light as compared with a controlgroup to which a physiological saline was administered whereby it isapparent that p 1c suppresses hypertrophy of heart and kidney.

TABLE 1 Weight of Organ (g/g body weiqht) Right Left SubstanceAdministered Heart Kidney Kidney Physiological saline 1 0.43 0.38 0.40 20.47 0.39 0.44 Mean Value 0.45 0.39 0.42 Standard Error 0.02 0.00 0.02 p1c 3 0.37 0.34 0.34 4 0.40 0.35 0.35 5 0.39 0.35 0.35 6 0.40 0.33 0.35Mean Value 0.39 0.34 0.35 Standard Error 0.01 0.00 0.00

Experimental Example 8

Pulmonary, artery, superior mesenteric artery and femoral artery wereexcised from the rats tested in Experimental Example 7 and their weightswere measured. Weight of each artery was calculated in terms of weightper each area, followed by further converting the calculated value intothe value per body weight of each rat and the results were compared andshown in Table 2. As shown in Table 2, weight of each artery in the SHRto which p 1c was administered was apparently light as compared withthat of the control group whereupon it has been found that p 1csuppresses the thickening of artery, i.e. remodeling of blood vessel.

TABLE 2 Weight of Artery (mg/mm²/g body weight) Superior PulmonaryMesenteric Femoral Substance Administered Artery Artery ArteryPhysiological saline 1 0.00093 0.00084 0.00138 2 0.00119 0.00107 0.00135Mean Value 0.00106 0.00096 0.00137 Standard Error 0.00013 0.000120.00002 p 1c 3 0.00067 0.00094 0.00068 4 0.00087 0.00095 0.00094 50.00088 0.00096 0.00106 6 0.00082 0.00100 0.00087 Mean Value 0.000810.00096 0.00089 Standard Error 0.00005 0.00001 0.00008

Example 3

A screening system was constructed using human prorenin preparedaccording to the description in Japanese Patent Laid-Open No.H10-279,600 and U.S. Pat. No. 5,945,512 and an antibody which is able toactivate human prorenin by specifically recognizing the human proreninpf peptide. A test was carried out using a screening system that wasconstructed for candidate compounds to confirm their action to theactivation of human prorenin by the anti-pf peptide antibody. By such amethod, substances can be obtained which are capable of inhibiting theactivation of human prorenin by anti-human pf peptide antibody. Inaddition, information that is accumulated on the basis of peptidesselected from human prorenin profragment region, known hypotensors, andthe like and also the result of the experiments using the animal modelcan make it possible to select substances having hypotensive effect,organ hypertrophy suppressing effect and artery thickening suppressingeffect from the resulting substances.

POSSIVILITY OF INDUSTRIAL USAGE

The present invention provides a method for the selection of a substancewhich is capable of controlling the activation of prorenin whereadjusting ability for activation of prorenin by protein-proteininteraction in a profragment region of prorenin is used as an indicatorand also provides a prorenin activation controlling substance having afunction of controlling the activation of prorenin based onprotein-protein interaction by a profragment region of prorenin. Inaddition, by the use of the above-mentioned prorenin activationcontrolling substance, it is possible to further provide hypotensor,organ hypertrophy suppressor and arterial thickening suppressor having anovel mechanism. When such a pharmaceutical agent is used either solelyor together with the already-known pharmaceutical agent, it can beeffectively utilized for the treatment of hypertension, cardiacinsufficiency, renal insufficiency, arteriosclerosis, the diseasesaccompanied by blood vessel remodeling and so on.

1. A method for selecting of a substance capable of controllingactivation of prorenin, said prorenin being non-proteolyticallyactivated to have renin-like activity by protein-protein interaction inthe profragment region of prorenin without alteration in a primarystructure, wherein said method comprising: a) providing prorenin, b)providing an antibody able to activate prorenin non-proteolytically tohave renin-like activity by interacting with the profragment region ofprorenin, c) providing a candidate substance, and d) measuring therenin-like activity of prorenin activated by the antibody provided instep (b) in the presence of the candidate substance, comparing saidactivity with the renin-like activity of prorenin activated by theantibody provided in the absence of the candidate substance, andselecting the candidate substance if there is an inhibition of therenin-like activity of prorenin in the presence of the candidatesubstance in comparison to the renin-like activity of prorenin in theabsence of the candidate substance.
 2. The method for selecting asubstance according to claim 1, wherein the protein-protein interactionis caused by an antibody against the profragment region of prorenin. 3.The method for selecting a substance according to claim 1, wherein thecandidate substance is a substance which is designed on the basis ofinformation of at least three amino acid sequences of the amino acidsequence in the profragment region of prorenin.
 4. The method forselecting a substance according to claim 3, wherein the basis ofinformation is contained in from the first to the 19th or from 27th tothe 41st amino acid sequence from N-terminal of the amino acid sequencein the profragment region of prorenin.
 5. The method for selecting asubstance according to claim 3, wherein the basis of information iscontained in from the 5th to the 19th amino acid sequence fromN-terminal of the amino acid sequence in the profragment region ofprorenin.
 6. The method for selecting a substance according to claim 3,wherein the substance is a peptide.
 7. The method for selecting asubstance according to claim 3, wherein the substance is a non-peptidelow-molecular weight compound.
 8. The method for selecting a substanceaccording to claim 1, wherein the prorenin is human prorenin.
 9. Themethod for selecting a substance according to claim 1, wherein theprorenin is rat prorenin.
 10. The method for selecting a substanceaccording to claim 2, wherein the substance is designed on the basis ofinformation of at least three amino acid sequences of the amino acidsequence in the profragment region of prorenin.
 11. The method forselecting a substance according to claim 10, wherein the substance is apeptide.
 12. The method for selecting a substance according to claim 10,wherein the substance is a non-peptide low-molecular weight compound.13. The method for selecting a substance according to claim 2, whereinthe prorenin is one of human prorenin and rat prorenin.
 14. The methodfor selecting a substance according to claim 3, wherein the prorenin isone of human prorenin and rat prorenin.
 15. The method for selecting asubstance according to claim 10, wherein the prorenin is one of humanprorenin and rat prorenin.
 16. The method for selecting a substanceaccording to claim 11, wherein the prorenin is one of human prorenin andrat prorenin.
 17. The method for selecting a substance according toclaim 12, wherein the prorenin is one of human prorenin and ratprorenin.
 18. A method for selecting a substance capable of controllingactivation of human prorenin, said human prorenin beingnon-proteolytically activated to have renin-like activity byprotein-protein interaction in the profragment region of human proreninwithout alteration in a primary structure, said method comprising a)providing prorenin of a non-human animal; b) providing an antibody ableto activate the prorenin of the non-human animal non-proteolytically tohave renin-like activity by interacting with the profragment region ofthe prorenin, wherein said antibody is obtained by using the informationof the partial sequence of an amino acid sequence in the prorenin of thenon-human animal corresponding to the partial sequence of an amino acidsequence in human prorenin involved in the activation of human prorenin;c) providing a candidate substance; d) measuring the renin-like activityof the prorenin of the non-human animal activated by the antibodyprovided in step (b) in the presence of the candidate substance andmeasuring the renin-like activity of the prerenin of the non-humananimal activated by the antibody in the absence of the candidatesubstance and selecting the candidate substance if there is aninhibition of the renin-like activity of the prerenin in the presence ofthe candidate substance in comparison to the renin-like activity of theprorenin in the absence of the candidate substance.